Amaç: Helicobacter pylori enfeksiyonlarının tanısında çeşitli
yöntem-ler kullanılmaktadır. Invazif olmayan, daha hızlı testyöntem-lerin kullanımı için artan bir talep vardır. Bu çalışmanın amacı farklı tanı yöntemle-rini karşılaştırmaktır.
Yöntemler: Çalışma özofagogastroduodenoskopi uygulanan 87
has-tayı kapsamaktadır. Bu hastalardan alınan biyopsi örneklerinden kültür, Real-time PCR ve histopatolojik inceleme yapıldı. Ayrıca tüm hastalardan dışkı örnekleri de alınıp H.pylori dışkı antijen testi (HpSA) uygulandı. Histopatolojik inceleme altın standart test olarak kabul edildi.
Bulgular: Histopatolojik inceleme sonucu; 87 hastanın 77’si H. pylori
pozitif (%87.5), 10’u ise (%12.5) negatif olarak belirlenmiştir. Kültür, HpSA ve Real-time PCR’nin duyarlılık ve özgüllükleri sırasıyla %71.4 ve %100, %87 ve %60, %97.4 ve % 80 olarak saptanmıştır. Kültür yönte-miyle pozitif saptanan 55 örneğin 48’inde antibiyotik duyarlılık testle-ri çalışılmıştır. Klatestle-ritromisin direnci 28 (% 58.3), metronidazol direnci 14 (% 29.2) ve levofloksasin direnci 4 (% 8.3) izolatta saptandı. Hiçbir izolatta amoksisilin ve tetrasiklin direnci saptanmadı.
Sonuç: Günümüzde, Helicobacter pylori enfeksiyonlarının tanısında
invazif olan ve invazif olmayan çeşitli tanı testleri kullanılmaktadır. Her testin bazı avantajları ve dezavantajları vardır. Seçilen tanı yönte-mi, tüm yaş gruplarına kolaylıkla uygulanabilir olmalıdır.
Anahtar Kelimeler: Antimikrobiyal direnç, tanı, Helicobacter pylori,
yöntemler, üst gastrointestinal kanama
Introduction: There are several methods used for the diagnosis of
He-licobacter pylori infections, and there is an increasing demand for the
use of non-invasive, more rapid tests. The aim of the present study was to compare different diagnostic methods.
Methods: A total of 87 patients who had undergone
esophagogastro-duodenoscopy were included in the study. Biopsy samples obtained from these patients were used for culture, real-time polymerase chain reaction (RT-PCR), and histopathological examination. Stool samples were also collected from these patients and were tested using the
Heli-cobacter pylori stool antigen (HpSA) kit. Histopathological examination
was accepted as the gold standard test.
Results: H. pylori was identified by histological examination in 77/87
(87.5%) patients, whereas it was negative in 10/87 (12.5%) patients. Furt-hermore, positive results were obtained in 55 (63.2%), 71 (81.6%), and 77 (87.5%) patients using the culture method, HpSA analysis, and RT-PCR met-hod, respectively. The sensitivity and specificity of culture, HpSA, and PCR tests were determined as 71.4% and 100%, 87% and 60%, and 97.4% and 80%, respectively. Antibiotic susceptibility tests were performed on 48 out of the 55 culture positive samples. Resistance to clarithromycin was found in 28 (58.3%), metronidazole in 14 (29.2%), and levofloxacin in 4 (8.3%) of the isolates. Resistance to amoxicillin and tetracycline was not observed.
Conclusion: There are currently several invasive and non-invasive
diag-nostic tests for the detection of H. pylori infections. Each test has some advantages and disadvantages. The diagnostic method of choice sho-uld be easy and applicable to all age groups.
Keywords: Antimicrobial resistance, diagnosis, Helicobacter pylori,
met-hods, upper gastrointestinal bleeding
Abstr
act / Öz
ORCID IDs of the authors: S.M.
9202-4047; A.A. 0000-0001-7856-7590; B.Ş. 0002-0724-3166; C.S. 0001-7637-8745; D.O. 0000-0003-3637-5392; H.Ö. 0000-0002-9063-3893; T.K. 0000-0003-2879-7739; Y.A. 0000-0002-0919-5508. This study was presented in XXXVII. Turkish Microbiology Congress (November 16-20, 2016, Antalya, Türkiye).
1Department of Medical Microbiology, Selçuk University School of Medicine, Konya, Türkiye
2Department of Medical Microbiology, Hacettepe School Faculty of Medicine, Ankara, Türkiye
3Department of Medical Pathology, Hacettepe School of Medicine, Ankara, Türkiye
4Departments of Pediatrics, Gastroenterology Unit, Hacettepe University School of Medicine, Ankara, Türkiye
5Departments of Internal Medicine, Gastroenterology Unit, Hacettepe University School of Medicine, Ankara, Türkiye
Address for Correspondence Yazışma Adresi:
Salih Maçin
E-mail: salihmacin@hotmail.com Received/Geliş Tarihi: 04.01.2017 Accepted/Kabul Tarihi: 22.11.2017 © Telif Hakkı 2018 Makale metnine istanbultipdergisi.org web sayfasından ulaşılabilir.
© Copyright 2018 by Available online at istanbulmedicaljournal.org
DOI: 10.5152/imj.2018.94834
Salih Maçin
1, Alpaslan Alp
2, Burçin Şener
2, Cenk Sökmensüer
3, Diclehan Orhan
3, Hasan Özen
4,
Taylan Kav
5, Yakut Akyön
2Methods
Patients
The present study was planned retrospectively. Data included 87 patients who had been evaluated at Departments of Pediatric Gas-troenterology and Internal Medicine, Hacettepe University School of Medicine. Biopsy specimens were obtained during esophago-gastroduodenoscopy. The study was conducted in accordance with the Declaration of Helsinki.
Culture
Samples were inoculated onto brain heart infusion (BHI; Oxoid, England) agar containing 7% horse blood and antibiotics (10 mg/L vancomycin, 5 mg/L trimethoprim, 5 mg/L cefsulodin, and 5 mg/L amphotericin B). All samples were incubated at 35°C-37°C for 5-7 days under microaerobic conditions. Bacterial isolates were identi-fied by Gram staining, colony morphology, and urease, catalase, and oxidase reactions (7).
Antibiotic Susceptibility Tests
Antibiotic susceptibility testing was performed using the gradient strip method. Amoxicillin, clarithromycin, tetracycline, levofloxa-cin, and metronidazole were tested. The identified strains were sub-cultured onto antibiotic-free 5% horse blood containing BHI agars and incubated at 35 °C for 3 days. Samples were inoculated onto Mueller-Hinton agar (Oxoid, England) containing 5% horse blood to perform antibiotic susceptibility testing. The European Committee on Antimicrobial Susceptibility Testing guidelines were used in order to evaluate the results (8). The H. pylori NCTC 11637 standard strain was used as the control strain.
Helicobacter pylori Stool Antigen (HpSA) Test
Stool samples obtained from the patients were stored at −20°C until use. Samples were tested for H. pylori antigen using the commer-cially available HpSA kit (GA Generic Assays GmbH, Germany). This is an indirect two-site immunoassay based on polyclonal antibodies, leading to qualitative determination of the antigen in stool samples.
Real-time (RT) PCR Method
DNA isolation was performed with MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Germany) in automated MagNa Pure nucleic acid isolation instrument. RT-PCR was per-formed in capillary tubes in the LightCycler 2.0 (Roche Diagnostics, Germany). Cycling conditions were denaturation at 95 °C for 10 min, followed by 40 cycles of amplification at 95°C for 10 s, 55°C for 10 s (with single acquisition of fluorescence), and 72°C for 15 s. Melting conditions were at 95°C for 10 s, 50 °C for 5 s, and 80°C for 0 s. Finally, a cooling step was applied at 40°C for 30 s (9).
Histopathological Examination
Two gastric biopsy specimens, one from the antrum and one from the corpus, were fixed in 10% formalin. Prepared sections (4 μm thickness) were placed on poly-l-lysine-coated adhesive micro-scope slides for immunohistochemical staining. All sections were first dewaxed (heating at 60°C in an autoclave) and then embed-ded in xylol for 10 min. Automatic immunohistochemical staining was performed using a Leica DS9800 system (New Castle, United Kingdom). Antigen retrieval was performed with citrate buffer (pH 6.0, 20 min, 95°C) for 10 min. Sections were incubated with H.
py-lori rabbit polyclonal antihuman antibody (215A-70; Cell Marque,
CA, USA) at a dilution of 1:100 for 1 h. A polymer detection kit (DS9800; New Castle, United Kingdom) was used to detect immu-nostaining. Sections were treated with diaminobenzidine as chro-mogen. H. pylori-infected tissues were used as positive controls. The primary antibody solutions were substituted with phosphate buffer solution in the negative staining controls.
Statistical Analysis
All the analysis was performed by SPSS (Statistical Packege for So-cial Sciences) for Windows version 15.0 (SPSS Inc.; Chicago, IL, USA). A p value ≤0.05 was considered statistically significant. Pearson correlation analysis was also conducted. P value was calculated using the McNemar test.
Results
The mean age of the patients was 17.8±10.6 years (min-max: 5-64 years), and 45 (51.7%) were men. Histopathological examina-tion identified 77/87 (87.5%) patients as positive, whereas 10/87 (12.5%) patients were negative. Positive results were obtained in 55 (63.2%), 71 (81.6%), and 77 (87.5%) patients by the culture method, HpSA analysis, and PCR method, respectively (Table 1).
Histopathological examination results were accepted as the gold standard, and specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for each meth-od (Table 2). The specificity of culture was 100%, and sensitivity was 71.4%. The sensitivity and specificity of HpSA and RT-PCR tests were found as 87% and 60% and 97.4% and 80%, respectively (Table 3). Histopathological examination results revealed that false-positive results were detected by HpSA in 5.6% (4/71) and by RT-PCR in 2.6% (2/77) of the patients. A higher rate of false-negative results was
Table 1. The evaluation of the diagnostic tests used for the detection of H. pylori in the study samples
Positive Negative
Method samples (%) samples (%) Total
Histopathology 77 (87.5) 10 (12.5) 87
Culture 55 (63.2) 32 (36.8) 87
HpSA 71 (81.6) 16 (18.4) 87
RT-PCR 77 (87.5) 10 (12.5) 87
HpSA: Helicobacter pylori stool antigen; RT-PCR: real-time polymerase chain reaction
Table 2. Comparison of the histopathology results with culture, HpSA, and RT-PCR results for the detection of H. pylori in the clinical specimens
Culture Culture HpSA HpSA RT-PCR RT-PCR
(+) (−) (+) (−) (+) (−) Histopathology 55 22 67 10 75 2 positive (n: 77) Histopathology 0 10 4 6 2 8 negative (n: 10) Total 55 32 71 16 77 10
HpSA: Helicobacter pylori stool antigen; RT-PCR: real-time polymerase chain reaction
obtained with the culture method (22/32; 68.7%). Culture method, RT-PCR, and HpSA tests were found to correlate with the Pearson correlation analysis (Table 4).
In 20 patients who had upper gastrointestinal bleeding, H. pylori was detected in all patients by RT-PCR, detected in 14 patients by culture, and detected in 16 patients by HpSA (Table 5).
Antibiotic susceptibility tests were performed on 48 out of the 55 culture positive samples. Among 48 isolates, none of them were resistant to amoxicillin and tetracycline. Resistance to clarithro-mycin was found in 28 (58.3%), metronidazole in 14 (29.2%), and levofloxacin in 4 (8.3%) of the isolates (Table 6).
Discussion
Currently, several different tests for the diagnosis of H. pylori in-fections exist. Each test has both advantages and disadvantages. Several studies have examined the diagnostic performance of in-vasive and non-inin-vasive methods (10, 11). However, these studies demonstrated a lack of agreement. Discrepancies in the diag-nostic performance of different tests in different studies might be attributed to the selection of different methods as the gold standard.
Among various diagnostic methods, histopathological examina-tion of endoscopic biopsy specimens provides more informaexamina-tion about the degree of inflammation and associated pathology (12). It is also the most reliable test in the presence of upper gastroin-testinal bleeding. Proton pump inhibitors should be stopped prior to gastroduodenoscopy, since they may decrease the sensitivity of the histopathological examination.
Routine cultivation is difficult to perform in microbiology labora-tories, since it is time consuming and hard to maintain microaero-philic conditions. However, bacterial growth in cultures provides definitive diagnosis and also enables antibiotic susceptibility test-ing to guide specific treatment. Gisbert and Abraria (13) reported three studies with culture sensitivity of 45% and specificity of 98%. Aktepe et al. (14) reported that the sensitivity of the culture meth-od is 61%, and specificity is 91%. In the present study, sensitivity was 71.4%, and specificity was 100% for culture. The low sensitivity and high specificity of the culture-based methods might be cor-related with inappropriate biopsy site and inadequate specimens. However, culture-based methods enable specific antibiotic suscep-tibility testing of the strains, thus providing important data espe-cially in populations with a high rate of drug resistance among H.
pylori strains.
HpSA test, which has been introduced as a non-invasive diagnos-tic alternative, has the advantages of being relatively inexpensive, easy to perform, and can be used in pregnant women, children, and the elderly. It can easily be performed in routine laborato-ries as it does not require complicated laboratory facilities. In a Japanese study, the sensitivity and specificity of the HpSA test were 93.9% and 95.7%, respectively, when compared with histopatho-logical examination (15). A study from Turkey reported the HpSA test sensitivity as 72% and specificity as 67% (14). In our study, sen-sitivity and specificity were 87% and 60%, respectively. There were four positive samples using the HpSA test. Two of these false-positive samples were negative using culture and RT-PCR. The two other samples were negative using histopathology and culture, but positive with RT-PCR method. Thus, this false-positive may be at-tributed to the lack of detection by histopathology that was con-sidered as the gold standard.
The presence of H. pylori and specific antibiotic-resistant genes can be investigated by RT-PCR from gastric biopsy specimens. RT-PCR has a high sensitivity and specificity and can be used as a follow-up assessment after therapy (16). The contamina-tion may occur during the DNA extraccontamina-tion step or the presence of inanimate microorganisms residual chromosomal DNA, and this may lead to false-positive results (17). In our study, biopsy PCR studies had a sensitivity of 97.4%, specificity of 80%, NPV
Table 5. Detection of H. pylori infection in patients with upper gastrointestinal bleeding
Positive samples Negative samples
Method (%) (%) Total
Histopathology 20 (100) 0 20
Culture 14 (70) 6 (30) 20
HpSA 16 (80) 4 (20) 20
RT-PCR 20 (100) 0 20
HpSA: Helicobacter pylori stool antigen; RT-PCR: real-time polymerase chain reaction Table 6. Antimicrobial resistance of the tested H. pylori strains (n=48) Antibiotics Resistance, n (%) Amoxicillin 0 Clarithromycin 28/48 (58.3%) Levofloxacin 4/48 (8.3%) Metronidazole 14/48 (29.2%) Tetracycline 0
Table 3. Comparison of the results of the H. pylori diagnostic methods when histopathology was considered as the gold standard
Sensitivity Specificity PPV NPV
Method (%) (%) (%) (%)
Culture 71.4 100 100 31.2
HpSA 87 60 94.4 37.5
RT-PCR 97.4 80 97.4 10.4
PPV: positive predictive value; NPV: negative predictive value; HpSA: Helicobacter pylori stool antigen; RT-PCR: real-time polymerase chain reaction Table 4. Correlation of H. pylori test results to
histopathology results
Method False-positive False-negative ra P
Culture 0 22 0.472 <0.0001
HpSA 4 10 0.387 0.180
RT-PCR 2 2 0.774 <1
aCorrelation is significant at the 0.01 level.
HpSA: Helicobacter pylori stool antigen; RT-PCR: real-time polymerase chain reaction
of 10.4%, and PPV of 97.4%. There were only two false-positive samples by RT-PCR. These two samples were also positive by HpSA test. Thus, obtaining positive results with two different methods provided strong information that this patient was in-fected with H. pylori, and the “false” false-positive evaluation in that specific case was attributed to choosing histopathology as the gold standard.
Many conventional H. pylori diagnostic methods show a significant decrease in their sensitivity in patients with upper gastrointestinal bleeding. However, in cases of upper gastrointestinal bleeding, PCR techniques and histopathological examination are more reli-able than rapid urease test, HpSA test, or culture (5, 18). A Korean study that evaluated the patients with gastrointestinal bleeding (n=157) found that sensitivity and specificity were 92.5% and 96% for histopathological examination, 40% and 100% for culture, and 97% and 56% for serology. The HpSA method showed relatively high sensitivity, but cannot be recommended as the primary diag-nostic method in the bleeding situation, because of its low speci-ficity (19). In our study, H. pylori was correctly detected by RT-PCR and histopathological examination in 20 patients who had upper gastrointestinal bleeding, culture in 14, and HpSA in 16. The find-ings of the current study suggested that histopathological exami-nation and RT-PCR assay were the most appropriate methods for the detection of H. pylori in patients with upper gastrointestinal bleeding.
Antibiotic susceptibility testing provides valuable information for choosing the right treatment. Resistance to metronidazole is re-ported to be between 15.9% and 77.9% (20, 21). In accordance with the literature, metronidazole resistance was 29.2% in the current study. Resistance to clarithromycin was reported to be 34% in Aus-tria, 54.6% in Spain, and 30.1% in Turkey (22-24). Clarithromycin resistance was also found to be high (58.3%) in the present study. Resistance to amoxicillin and tetracycline is very rare. Agudo et al. (23) revealed no resistance to amoxicillin, rifampicin, and tetra-cycline. Vécsei et al. (20) reported tetracycline resistance as 0.9%; however, they did not report any amoxicillin resistance. In our study, no resistance to amoxicillin and tetracycline was found. The frequency of levofloxacin resistance is reported to be 5.9%-18.2% in Turkey, 22.1% in Italy, and 34.5% in China (25-28). In our study, levofloxacin resistance was also found to be compatible with previ-ous studies from Turkey (8.3%).
Conclusion
There are a variety of tests available for the diagnosis of H. pylori. In cases where endoscopy could not be performed, non-invasive, simple, rapid, and practical HpSA test with acceptable results can be used to provide diagnosis and to monitor treatment. RT-PCR method has a high sensitivity and specificity in comparison with histopathological examination accepted as the gold standard. Both RT-PCR and histopathological examination are also reliable diagnostic methods in patients with upper gastrointestinal bleed-ing. When available, culture should be performed for antibiotic susceptibility tests, especially in the case of treatment failure. Thus, each laboratory should better establish its own diagnostic algorithm for the accurate and appropriate diagnosis of H. pylori infection in their patient population, according to their laboratory facilities and clinical setting.
Ethics Committee Approval: Since our study was retrospective and we
compared the tests previously used for Helicobacter pylori in our hospital, no ethics committee approval was applied.
Informed Consent: Informed consent is not obtained due to the
retrospec-tive nature of this study.
Peer-review: Externally peer-reviewed.
Author contributions: Concept - S.M, Y.A; Design -S.M, A.A, B.Ş., Y.A.;
Su-pervision - Y.A, B.Ş, H.Ö, T.K; Materials - C.S., D.O., SM; Data Collection and/ or Processing - A.A., B.Ş., C.S., D.O.; Analysis and/or Interpretation - S.M., Y.A.; Literature Review - S.M., B.Ş., Y.A.; Writing - S.M., A.A., B.Ş., C.S., D.O., H.O., T.K., Y.A.; Critical Review - A.A., B.Ş., Y.A.
Conflict of Interest: Authors have no conflicts of interest to declare. Financial Disclosure: The authors declared that this study has received no
financial support
Etik Komite Onayı: Çalışmamız retrospektif olduğu ve hastanemizde daha
önceden Helicobacter pylori tanısında kullanılan testler karşılaştırıldığı için, etik komite onayı başvurusu yapılmamıştır.
Hasta Onamı: Bu çalışma retrospektif veriler kullanılarak yapıldığından
hasta onamı alınmamıştır.
Hakem Değerlendirmesi: Dış Bağımsız.
Yazar Katkıları: Fikir - S.M, Y.A; Tasarım - S.M, A.A, B.Ş., Y.A.; Denetleme
- Y.A, B.Ş, H.Ö, T.K; Gereçler - C.S., D.O., S.M.; Veri Toplanmasi ve/veya İşle-mesi - A.A., B.Ş., C.S., D.O.; Analiz ve/veya Yorum - S.M., Y.A.; Literatur Tara-masi - S.M., B.Ş., Y.A.; Yaziyi Yazan - S.M., A.A., B.Ş., C.S., D.O., H.O., T.K.,Y.A..; Elestirel Inceleme - A.A., B.Ş., Y.A.
Çıkar Çatışması: Yazarların beyan edecek çıkar çatışması yoktur. Finansal Destek: Yazarlar bu çalışma için finansal destek almadıklarını
beyan etmişlerdir.
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